Nghiên cứu khả năng tạo Bacteiocin của vi khuẩn Lactobacillus acidphilus.pdf
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BỘ Y TẾ TRƢỜNG ĐẠI HỌC DƢỢC HÀ NỘI PHẠM NGUYÊN PHƢƠNG NGHIÊN CỨU KHẢ NĂNG TẠO BACTERIOCIN CỦA VI KHUẨN LACTOBACILLUS ACIDOPHILUS KHÓA LUẬN TỐT NGHIỆP DƢỢC SĨ HÀ NỘI – 2013
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Transcript of Nghiên cứu khả năng tạo Bacteiocin của vi khuẩn Lactobacillus acidphilus.pdf
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B Y T TRNG I HC DC H NI
PHM NGUYN PHNG
NGHIN CU KH NNG TO
BACTERIOCIN CA VI KHUN
LACTOBACILLUS ACIDOPHILUS
KHA LUN TT NGHIP DC S
H NI 2013
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B Y T TRNG I HC DC H NI
PHM NGUYN PHNG
NGHIN CU KH NNG TO
BACTERIOCIN CA VI KHUN
LACTOBACILLUS ACIDOPHILUS
KHA LUN TT NGHIP DC S
Ngi hng dn: DS. L Ngc Khnh Ni thc hin: B mn Cng nghip Dc
H NI 2013
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LI CM N
Trc ht, em xin gi li cm n su sc ti DS. L Ngc Khnh, ngi
trc tip hng dn v to mi iu kin thun li em c th hon
thnh kha lun ny.
Em xin chn thnh cm n TS. m Thanh Xun ng gp nhiu
kin qu bu v tn tnh gip em thc hin ti.
Em cng xin cm n s quan tm, gip ca cc thy c gio, cc anh
ch k thut vin B mn Cng nghip Dc i vi em trong sut qu trnh
nghin cu v lm thc nghim ti b mn.
Cui cng, em xin by t lng bit n gia nh, thy c, bn b, nhng
ngi lun ng vin, gip em trong hc tp v trong cuc sng.
Em xin trn trng cm n!
H Ni, ngy 17 thng 5 nm 2013
Sinh vin
Phm Nguyn Phng
-
MC LC
Danh mc cc k hiu, cc ch vit tt
Danh mc cc bng
Danh mc cc hnh v, th
T VN 1
Chng 1. TNG QUAN 3
1.1. Khi qut v bacteriocin 3
1.1.1. Vi nt v lch s 3
1.1.2. Phn b 3
1.1.3. Danh php v phn loi 3
1.1.4. Tnh hnh nghin cu 4
1.2. Bacteriocin ca vi khun lactic 6
1.2.1. Vi nt v vi khun lactic sinh bacteriocin 6
1.2.2. Phn loi 7
1.2.3. Tnh cht 8
1.2.4. Sinh tng hp, ph tc dng v c ch tc dng 9
1.2.5. nh gi hot tnh bacteriocin 11
1.2.6. Chit xut v tinh ch 11
1.2.7. ng dng 13
Chng 2. NGUYN LIU, THIT B, NI DUNG V PHNG
PHP NGHIN CU
16
2.1. Nguyn vt liu, thit b 16
2.1.1. Chng vi khun 16
2.1.2. Ha cht 16
2.1.3. Mi trng 16
2.1.4. My mc, dng c 17
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2.2. Ni dung nghin cu 18
2.2.1. S b xc nh bacteriocin trong dch ln men v sinh khi vi
khun L. acidophilus ATCC 4653
18
2.2.2. Nghin cu mt s tnh cht ca bacteriocin sinh ra bi vi khun
L. acidophilus ATCC 4653
18
2.3. Phng php nghin cu 18
2.3.1. Phng php bo qun ging vi sinh vt 18
2.3.2. Phng php nhn ging L. acidophilus 19
2.3.3. Phng php x l sinh khi L. acidophilus 20
2.3.4. Phng php nh gi hot tnh bacteriocin theo c ch khuch
tn qua ging thch
20
2.3.5. Phng php kho st nh hng ca pH n hot tnh
bacteriocin
21
2.3.6. Phng php kho st nh hng ca nhit n hot tnh
bacteriocin
22
2.3.7. Phng php kho st kh nng chit bacteriocin bng
(NH4)2SO4
22
Chng 3. THC NGHIM, KT QU V BN LUN 23
3.1. S b xc nh bacteriocin trong dch ln men v sinh khi vi
khun L. acidophilus ATCC 4653
23
3.1.1. Xc nh bacteriocin trong dch ln men 23
3.1.2. Xc nh bacteriocin trong sinh khi 26
3.2. Nghin cu mt s tnh cht ca bacteriocin sinh ra bi vi
khun L. acidophilus ATCC 4653
28
3.2.1. Nghin cu nh hng ca pH n hot tnh bacteriocin ca L.
acidophilus ATCC 4653
28
3.2.2. Nghin cu nh hng ca nhit n hot tnh bacteriocin ca 31
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L. acidophilus ATCC 4653
3.2.3. Nghin cu kh nng chit bacteriocin ca L. acidophilus ATCC
4653 bng phng php kt ta (NH4)2SO4
34
KT LUN V KIN NGH 39
TI LIU THAM KHO
PH LC
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DANH MC CC K HIU, CC CH VIT TT
ATCC (American Type Culture
Collection)
Bo tng ging chun vi sinh vt Hoa
K
BC (Bacterial Cellulose) Cellulose vi khun
Dha Dehydroalanine
Dhb Dehydrobutyrine
FDA (U.S. Food and Drug
Administration)
Cc qun l Thc phm v Dc
phm Hoa K
GC Guanine v Cytosine
GRAS (Generally Recognized As
Safe)
c cng nhn chung l an ton
kDa kiloDalton
LAB (Lactic Acid Bacteria) Vi khun lactic
Lan Lanthionine
MeLan MethylLanthionine
MRS de Man, Rogosa, Sharpe
MRSA (Methicillin-Resistant
Staphylococcus aureus)
T cu vng khng methicillin
PMF (Proton Motive Force) ng lc proton
SDS (Sodium Dodecyl sulfate) Natri dodecyl sulfat
sp. (species) Loi
subsp. (subspecies) Phn loi
VRE (Vancomycin-Resistant
Enterococci)
Cu khun rut khng vancomycin
UV (Ultraviolet) Tia cc tm
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DANH MC CC BNG
STT Tn bng Trang
1.1 Phn loi bacteriocin ca LAB 8
2.1 Cc ha cht s dng trong ti 16
2.2 Cc my mc s dng trong ti 17
3.1 Kt qu xc nh bacteriocin trong dch ln men ca L.
acidophilus ATCC 4653
24
3.2 Kt qu xc nh bacteriocin trong sinh khi ca L.
acidophilus ATCC 4653
26
3.3 Hot tnh bacteriocin ca L. acidophilus ATCC 4653 cc
gi tr pH nghin cu trn vi khun kim nh B. subtilis
ATCC 6633
30
3.4 Hot tnh bacteriocin ca L. acidophilus ATCC 4653 sau
khi c x l nhit trn vi khun kim nh B. subtilis
ATCC 6633
32
3.5 Hot tnh bacteriocin ca phn ta v phn dch thu c
khi b sung (NH4)2SO4 vo dch ln men ca L.
acidophilus ATCC 4653 vi vi khun kim nh l B.
subtilis ATCC 6633
35
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DANH MC CC HNH V, TH
STT Tn hnh Trang
1.1 Biu thng k s lng cc cng b lin quan n
bacteriocin trn Pubmed trong giai on t 1950 2010
5
3.1 Vng c ch to bi dch ln men ca L. acidophilus ATCC
4653 trn vi khun kim nh B. subtilis ATCC 6633
26
3.2 Vng c ch to bi dch ln men ca L. acidophilus ATCC
4653 trn vi khun kim nh E. coli ATCC 25922
26
3.3 nh hng ca pH n hot tnh bacteriocin ca L.
acidophilus ATCC 4653
31
3.4 nh hng ca nhit n hot tnh bacteriocin ca L.
acidophilus ATCC 4653
33
3.5 S thay i hot tnh bacteriocin ca phn ta v phn dch
theo nng (NH4)2SO4 s dng
36
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T VN
Vic pht hin ra cc cht khng sinh nh du mt bc tin cch mng
ca loi ngi trong cuc chin chng li cc bnh nhim khun, cng ngh
khng sinh c nh gi l mt trong nhng thnh tu khoa hc ln nht ca
th k XX [52]. Tuy nhin, bc vo th k XXI, con ngi ang phi i mt
vi k nguyn hu khng sinh vi s thiu ht cc nhm khng sinh mi trong
khi cc chng vi khun khng thuc khng ngng gia tng [13]. Trc tnh
hnh , vic nghin cu v pht trin nhng hp cht mi c tc dng khng
khun gim s ph thuc vo khng sinh l ht sc cn thit. Bacteriocin
chnh l mt trong s [52].
Bacteriocin l nhng peptid hay protein c tc dng dit khun do vi
khun sinh ra [34]. Chng c pht hin t u th k XX nhng mi
c nghin cu rng ri trong mt vi thp nin tr li y khi nhu cu v
nhng hp cht khng khun nhm thay th khng sinh v cc cht bo qun
ha hc tr nn cp thit [26]. Vi c tnh an ton c FDA xc nhn, vi
khun lactic (LAB) l mt nhm vi khun sinh bacteriocin thu ht s quan
tm c bit ca cc nh khoa hc [52]. Nhiu cng trnh nghin cu
chng minh tc dng c ch vi khun c hi ca cc bacteriocin sinh ra bi
LAB [20]. Nisin mt bacteriocin ca Lactococcus lactis spp. c s
dng rng ri trn th gii nh mt cht bo qun ngun gc sinh hc an ton
v hiu qu [23]. Thnh cng ca nisin bc u cho thy tim nng ng
dng to ln ca bacteriocin t LAB [22].
Ti Vit Nam, bacteriocin ca LAB bt u c nghin cu trong
nhng nm gn y, tuy nhin s lng nghin cu cha nhiu v tp trung
ch yu trong nhm ngnh thc phm v th y. Vi mong mun gp phn
nh vo nghin cu bacteriocin ca LAB Vit Nam, ti Nghin cu
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kh nng to bacteriocin ca vi khun Lactobacillus acidophilus c
thc hin vi cc mc tiu c th nh sau:
1. S b xc nh bacteriocin trong dch ln men v sinh khi vi khun L.
acidophilus ATCC 4653.
2. Nghin cu mt s tnh cht ca bacteriocin sinh ra bi L. acidophilus
ATCC 4653.
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Chng 1. TNG QUAN
1.1. Khi qut v bacteriocin
1.1.1. Vi nt v lch s
Nhng bo co u tin lin quan ti bacteriocin c cho l c t cui
th k XIX khi cc nh khoa hc nghin cu v tnh i khng gia cc vi
khun (bacterial antagonism) [26]. Nm 1925, bacteriocin chnh thc c
Gratia tm ra trn mt chng Escherichia coli [74], v sau cht ny c bit
n vi tn gi colicin V [73] v nay l microcin V [28]. Nm 1953, thut
ng bacteriocin ln u c xut v nh ngha bi Jacob v cng s,
ch yu da trn cc c tnh ca colicin [34]. Nm 1976, Tagg v cng s
ch ra s khc bit gia colicin v cc bacteriocin sinh ra bi vi khun Gram
(+) [66], t nh ngha v bacteriocin c m rng v n ngy nay, cc
peptid v protein do vi khun sinh ra c tc dng dit khun u c gi l
bacteriocin [34].
1.1.2. Phn b
Bacteriocin l sn phm ph bin ca gii vi khun [34]. Chng c
tm thy nhiu nhm vi khun tht (Eubacteria) v c trn vi khun c
(Archaebacteria) [59]. n nay, c khong 200 bacteriocin c m t
[26]. Tuy nhin s lng bacteriocin trong thc t c c tnh ln hn th
rt nhiu. Theo Klaenhammer, 99% cc loi vi khun c th sinh ra t nht
mt bacteriocin [41].
1.1.3. Danh php v phn loi
a. Danh php
Hin nay cha c h thng danh php quc t cho bacteriocin. Tn ca
bacteriocin thng xut pht t tn chi hay tn loi vi sinh vt u tin c
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pht hin sinh ra bacteriocin . V d nh colicin t E. coli, pyocin t
Pseudomonas pyocynea, megacin t Bacillus megaterium [25]
b. Phn loi
H thng phn loi quc t dnh cho bacteriocin cha c chnh thc
xy dng v hin vn ang l ch tranh lun trong gii khoa hc [26].
Riley v Wertz nghin cu bacteriocin theo 3 nhm: bacteriocin ca vi khun
Gram (-), bacteriocin ca vi khun Gram (+) v bacteriocin ca vi khun c
[59]. Chi tit hn, theo Desriac v cng s, bacteriocin c phn loi cn c
nhiu tiu ch, trong cc tiu ch c bn bao gm: h vi khun sinh
bacteriocin, trng lng phn t, trnh t chui acid amin v cu trc ca
on gen (gene cluster). Cc tc gi chia bacteriocin lm 2 nhm ln: nhm
th nht l cc bacteriocin bn cht protein (protein-bacteriocin), thng c
sinh ra bi vi khun Gram (-), tiu biu l cc vi khun ng rut
(Enterobacteriae); nhm th 2 l cc bacteriocin bn cht peptid (peptid-
bacteriocin), thng c sinh ra bi vi khun Gram (+), tiu biu l cc vi
khun lactic (LAB). Mi nhm li c chia thnh nhiu lp v phn lp [26]
(Ph lc 1).
1.1.4. Tnh hnh nghin cu
a. Th gii
D c tm ra t kh sm (1925) nhng thi gian u s lng cc
nghin cu v bacteriocin khng nhiu v hu ht tp trung vo cc
bacteriocin ca vi khun Gram (-). Cho n nhng nm 60 ca th k XX, mi
ch c 3 chi vi khun Gram (+) c i su nghin cu v kh nng sinh
bacteriocin l Bacillus, Listeria v Staphylococcus [26]. Tuy nhin, cng
trong giai on ny, nisin v subtilin 2 bacteriocin quan trng t vi khun
Gram (+) c pht hin. Roger v cng s cng b nghin cu u tin
v nisin vo nm 1928 [60], trong khi subtilin c Jansen v Hirschmann
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pht hin vo nm 1944 t vi khun Bacillus subtilis [37]. n u nhng
nm 70, trnh t chui acid amin ca 2 bacteriocin ny mi c xc nh r
[29] [75].
K t nhng nm 80, s lng cc nghin cu v bacteriocin c s gia
tng ng k, bao gm c cc nghin cu trn colicin v trn cc bacteriocin
khc [26]. Tip , vic nisin c FDA cng nhn t tiu chun GRAS vo
nm 1988 [58] thc y s quan tm ca cc nh khoa hc i vi cc
bacteriocin ca LAB bi tim nng ng dng to ln trong cng ngh thc
phm, y t, th y... Kt qu tt yu l s bng n cc nghin cu v
bacteriocin ca LAB vo nhng thp nin cui th k XX v u th k XXI
[26] (Hnh 1.1).
Hnh 1.1: Biu thng k s lng cc cng b lin quan n bacteriocin
trn Pubmed trong giai on t 1950 2010 [26]
Trong 20 nm gn y, trn ton th gii c 706 bng sng ch lin quan
ti bacteriocin ca LAB c cp, trong s c 421 bng trong lnh vc
cng ngh bo qun thc phm v 124 bng v cht tr sinh (probiotic) cho
ng vt [26]. Cc kh nng ng dng khc ca bacteriocin nh ngn nga
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6
nhim trng khu tr [64], lm cht bo v thc vt [33], lm cht thay th
khng sinh [38] cng c xut v nghin cu bc u.
Microcin v bacteriocin t vi khun bin (marine bacteria) l 2 nhm
bacteriocin mi c a ra gn y [26]. Microcin c ngun gc t vi khun
ng rut nh colicin [28] nhng khi lng phn t nh hn v phn ln
c bin i sau dch m, tng t nhm lantibiotic vi khun Gram (+)
[35]. Cc bacteriocin t vi khun bin n nay vn cha c nghin cu
nhiu tuy nhin c d bo l s pht trin mnh trong tng lai gn [26].
b. Vit Nam
Vit Nam, bacteriocin bt u c cc nh khoa hc quan tm nghin
cu trong nhng nm gn y, c bit l bacteriocin ca LAB [8]. Bn cnh
cc nghin cu l thuyt [1] [2] [8], mt s cng trnh bc u a
bacteriocin vo ng dng trong thc tin. Nm 2008, cc tc gi Nguyn
Thy Hng v Trn Th Tng An s dng mng mng BC hp ph dch
bacteriocin ca Lactococcus lactis bo qun tht ti s ch ti thiu, gip
tng thi gian bo qun t 1 ln 3 ngy [3]. Vic ng dng cc k thut tin
tin nh c nh t bo [3], ti t hp gen [6] [7] nhm mc tiu sn xut
bacteriocin t hiu qu cao cng ang c nghin cu.
1.2. Bacteriocin ca vi khun lactic
1.2.1. Vi nt v vi khun lactic sinh bacteriocin
Vi khun lactic (LAB) c nh ngha l mt nhm vi khun Gram (+),
gm cc trc khun v cu khun khng sinh bo t, khng a kh (non-
aerobic) nhng chu oxy (aerotolerant), sinh ra acid lactic l sn phm chnh
khi ln men carbohydrat. LAB gm nhiu chi, trong tiu biu nht l cc
chi Lactobacillus, Leuconostoc, Pediococcus v Streptococcus [43].
Cc LAB u khng di ng, catalase m tnh, t l GC thp di 50%
[61]. LAB a m v chu nhit, nhit sinh trng ti u ty tng loi, dao
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ng t 28 n 45oC. LAB c nhu cu dinh dng phc tp, khi ln men cn
cc vitamin, acid amin v peptid ngn. Ngun carbohydrat c th l glucose,
fructose, lactose, maltose, sucrose; mt s chng vi khun c th s dng tinh
bt nh Lactobacillus amylophilus v L. amylovorus [5].
LAB phn b rng ri trong nhiu h sinh thi, thng c tm thy
trong mt s loi thc phm (cc sn phm t sa, tht v rau qu ln men,
bt nho chua), trn thc vt, trong ng tiu ha, h hp v sinh dc ca
ngi v ng vt [61].
LAB c lch s ng dng lu i trong thc phm ln men v cc tc
ng c li v mt dinh dng, cm quan v tui th sn phm, nht l vi
cc sn phm d hng nh sa, tht, c, mt s loi rau qu [43] [69]. Trong y
dc, nhiu loi LAB c s dng lm probiotic ci thin v nng cao
sc khe vi nhiu tc dng tr liu ng ch nh ngn nga nhim khun
ng rut, iu tr tiu chy, tng cng p ng min dch, gim
cholesterol mu, gim nguy c mc ung th [61]
Tc dng khng khun l mt c im rt ng ch ca LAB. LAB
sinh ra nhiu loi cht c tc dng khng khun: acid lactic, acid acetic,
ethanol, acid bo, H2O2, diacetyl v c bit l bacteriocin. Mc d
bacteriocin c th c tm thy nhiu loi vi khun khc, bacteriocin sinh
ra bi LAB vn thu ht c s ch c bit ca cc nh khoa hc bi tnh
an ton v tc dng c ch nhiu vi khun gy bnh nguy him nh
Staphylococcus aureus, Listeria monocytogenes [20] Trong s khong 200
bacteriocin c m t cho n nay, bacteriocin t LAB chim ti 90%
[26].
1.2.2. Phn loi
Bacteriocin sinh ra bi LAB rt a dng, trong vic phn loi chng
cng tn ti nhiu quan im khc nhau. Cch phn loi ph bin nht do
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Klaenhammer v cng s xut vo nm 1993 [26], chia bacteriocin ca vi
khun Gram (+) ni chung, ca LAB ni ring lm 3 lp [42]. c im ca 3
lp ny c tm tt trong bng 1.1. Mt s tc gi xut lp th 4 gm
cc bacteriocin phc tp trong cu trc c carbohydrat hoc lipid; tuy nhin
d liu v cc bacteriocin loi ny cn rt hn ch nn cn c nghin cu
thm [34].
Bng 1.1: Phn loi bacteriocin ca LAB [26] [34] [71]
Lp c im V d
I - Cn gi l cc lantibiotic
- Peptid nh (thng < 5 kDa)
- Bn nhit
- Bin i mnh sau dch m, dn ti s to thnh
cc acid amin him gp: Lan, MeLan, Dha, Dhb
- Chia lm 2 typ: typ A v typ B
Nisin, lactocin S,
epidermin
II - Peptid nh (thng < 10 kDa)
- Bn nhit
- t bin i sau dch m, khng cha Lan
- Chia lm 4 phn lp: IIa, IIb, IIc, IId
Pediocin PA-1,
lactacin F,
enterocin AS-
48
III - Protein kch thc ln (thng > 30 kDa)
- Km bn nhit
Helveticin J,
millericin,
enterolysin
1.2.3. Tnh cht
Tr mt s t cc bacteriocin thuc lp III l cc protein, bacteriocin ca
LAB a phn l cc peptid [26]. Chng thng mang in (+), c tnh s
nc (hydrophobic) hoc lng thn (amphiphilic) nhng a phn l lng
thn, gm t 20 n 60 acid amin [50] [71]. Ging nh nhiu bacteriocin ca
vi khun Gram (+), chng c th tn ti di dng cc kt tp c khi lng
ln (30 300 kDa) [54] [66]. Dng kt tp lm gim hot tnh ca
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bacteriocin, ng thi d gy nhm ln trong vic xc nh khi lng phn
t ca bacteriocin tinh khit. ph v cc kt tp ny, c th s dng cc
tc nhn phn tch nh ur, SDS, cc bin php nh siu lc, chit loi lipid
bng dung mi hu c [54].
n nh l mt tiu ch quan trng khi nghin cu v bacteriocin, n
lin quan ti kh nng sn xut v a bacteriocin vo ng dng trong thc
t. 3 yu t nh hng thng c nh gi l nhit , pH v enzym [54];
nh hng ca cc yu t ny vi mi bacteriocin c th rt khc nhau [66].
a s bacteriocin ca LAB kh bn nhit (tr lp III), chu pH acid tt hn
pH kim [66], d b bt hot bi cc enzym phn gii protein nh proteinase
K, pronase, pepsin, trypsin [54].
Ngoi mt s nt chung, cc bacteriocin ca LAB c nhng c im
ring rt a dng. Mt s tnh cht c bn ca cc bacteriocin tiu biu t
LAB c gii thiu trong Ph lc 2.
1.2.4. Sinh tng hp, ph tc dng v c ch tc dng
a. Sinh tng hp
Bacteriocin c tng hp ti ribosom di dng tin cht khng hot
ng, chng ch tr nn c hot tnh sau khi c bin i nh cc phn ng
enzym c hiu [51]. Theo Tagg v cng s, cc bacteriocin ca vi khun
Gram (+) c th tn ti di dng ngoi bo (extracellular form) hoc gn vi
t bo (cell-associated form), t l mi dng ph thuc vo c im mi
trng [66].
Mi trng sng ca vi khun cn c nh hng ln ti lng
bacteriocin sinh ra, trong cc yu t ch yu l thnh phn mi trng,
nhit v pH. Cc yu t ny c th nh hng trc tip n s sn sinh
bacteriocin hoc gin tip thng qua s sn xut sinh khi do bacteriocin
c coi l sn phm trao i cht bc 1 ca vi khun [20].
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10
b. Ph tc dng
Ph tc dng ca cc bacteriocin nhn chung rt khc nhau (Ph lc 2).
Cn c ph tc dng, c th chia bacteriocin ca LAB lm 2 nhm. Nhm th
nht l cc bacteriocin ch c tc dng trn cc loi gn gi vi loi sinh ra
n, v d nh lactobin A sinh ra bi Lactobacillus amylovorus ch c ch mt
s Lactobacillus sp. gn gi. Nhm th 2, t gp hn, c ph tc dng rng, v
d nh nisin sinh ra bi mt s dng Lactococcus lactis subsp. lactis c kh
nng c ch nhiu vi khun Gram (+). Ni chung, cc bacteriocin ca LAB
thng t th hin hot tnh trn vi khun Gram (-) [54].
Bacteriocin c th c tc dng dit khun hoc km khun trn cc chng
vi khun nhy cm, mc tc dng khc nhau do nh hng bi nhiu yu
t nh nng , mc tinh sch bacteriocin, cc iu kin thc nghim
[17]
c. C ch tc dng
C ch tc dng ca bacteriocin rt a dng, mt bacteriocin c th c
nhiu hn mt c ch tn cng t bo ch [52]. Mt s c ch c
xut: thay i hot tnh enzym ni bo, c ch bo t ny mm, to l trn
mng t bo Nhn chung, lp v t bo c coi l ch tc dng chnh ca
cc bacteriocin t LAB. Bacteriocin ph v tnh ton vn ca lp v t bo vi
khun nhy cm, gy mt cc cht ni bo, tiu hao PMF, cui cng gy cht
t bo [54].
C ch tc dng c th ca bacteriocin tng lp l khc nhau. Cc
bacteriocin lp I (lantibiotic) c ch tng hp vch t bo v / hoc to l trn
mng t bo bng cch gn vo lipid II [52]. Cc bacteriocin lp II c cu trc
xon c v lng thn nn c th xen vo mng t bo ch, t to l, kh
cc v lm cht t bo [18]. C ch tc dng ca cc bacteriocin lp III ni
chung cha r [54]; mt s tc gi cho rng l cc lysin tiu khun hay tiu
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11
khun t (bacteriolysin), tc ng trc tip ln vch t bo ch gy ph hy
t bo [18].
1.2.5. nh gi hot tnh bacteriocin
C nhiu phng php nh gi hot tnh bacteriocin, v c bn chng
u da trn vic nh gi tc dng i khng ca mu th i vi cc chng
vi sinh vt ch th [66]. Mt s v d: phng php spot-on-lawn, phng
php khuch tn qua ging thch [56], phng php o c [40] (Ph
lc 3). nh hng ca cc tc nhn c ch khc khng phi l bacteriocin nh
pH, H2O2 cn c loi tr bng phng php thch hp [66].
Gn y mt s phng php mi nh gi hot tnh bacteriocin
c xut. Nm 2001, Mugochi v cng s a ra phng php nh gi
hot tnh bacteriocin bng cch o nng ion K+ gii phng t vi khun ch
th [48]. Phng php ny c cho l c hiu qu snh ngang vi phng
php khuch tn qua ging thch [34].
1.2.6. Chit xut v tinh ch
Do tnh cht ca cc bacteriocin rt a dng nn khng c phng php
hay qui trnh chit tch chung [32]. Vn thng gp trong qu trnh chit
xut v tinh ch bacteriocin l s hao ht bacteriocin v s gim hot tnh c
ch vi sinh vt [66]. Mt qui trnh sn xut bacteriocin ch c coi l l tng
khi c th p dng vi qui m ln, cho hiu sut thu nhn bacteriocin trn
50% v tinh sch khong 90% [54].
a. Chit xut
Bacteriocin c th c chit xut bng nhiu cch, tiu biu trong s
l cc phng php: kt ta bng amoni sulfat, hp ph - gii hp ph v chit
bng dung mi hu c [56].
- Kt ta bng amoni sulfat
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12
Do c bn cht protein, bacteriocin c th c kt ta bng mui do
hin tng salting out (tnh tan ca protein gim mnh nng mui cao)
[56]. Amoni sulfat thng c s dng nht [56], ngoi ra cng c tc gi s
dng diatomite calcium silicate [19]. Mui c thm t t vo mu (dch ni
loi sinh khi t bo) cho n khi t nng mong mun. Nng mui
cn thit kt ta cc bacteriocin khc nhau c th khc nhau, cn kho st
tm ra nng mui thch hp vi tng trng hp c th. Ta bacteriocin
c tch ring bng cch li tm, sau ho trong lng nh (va ) m
phosphat pH 7 (gi tr pH ca dung dch m c th khc nhau gia cc
nghin cu) hoc nc ct v trng. Thng loi mui bng bng phng
php thm tch [56].
- Hp ph - gii hp ph
Phng php ny c pht trin bi Yang v cng s da trn c tnh
ca mt s bacteriocin: bacteriocin c hp ph ln b mt t bo vi khun
sinh bacteriocin pH gn trung tnh v c gii phng tr li mi trng
sau khi x l pH thp (pH khong 1,5 2,0) [70]. u im ca phng php
ny l t ko theo cc protein tp so vi phng php kt ta bng amoni
sulfat [56].
- Chit bng dung mi hu c
Phng php ny cng xut pht t bn cht protein ca bacteriocin: khi
thm dung mi hu c vo mu, hng s in mi gim xung, tan ca
protein gim v to kt ta [4]. Ethanol v aceton l hai dung mi kh thng
dng c s dng chit bacteriocin. Phng php chit bng dung mi
hu c c ng dng thnh cng vi mt s bacteriocin nh lactococcin
B, pediocin PA-1, lacticin Q [56]
b. Tinh ch
-
13
Bacteriocin sau khi c chit xut thng cn ln nhiu tp. thu
c bacteriocin tinh khit cn tri qua cc bc tinh ch [54]. Cc k thut
tinh ch bacteriocin rt a dng (Ph lc 4), c th da trn kch thc (v d:
sc k lc gel, siu lc) hoc in tch ring ca bacteriocin (v d: sc k
trao i ion, in di) [66]. Qu trnh tinh ch thng tri qua nhiu bc,
kt hp nhiu k thut khc nhau [54]. Tuy nhin, mt s qui trnh n gin
nhm tinh ch bacteriocin qui m cng nghip c pht trin thnh
cng. Nm 1997, Surez v cng s xut qui trnh tinh ch nisin A bng
phng php sc k i lc min dch nh khng th n dng khng nisin A,
cho kt qu tt [63]. Nm 2004, Cheigh v cng s tinh ch c nisin Z
t mi trng nui cy Lactococcus lactis subsp.lactis A164 bng phng
php sc k trao i ion nn m rng, thu c nisin Z c tinh khit gp 31
ln vi hiu sut 90% [16].
1.2.7. ng dng
Vi tc dng khng khun, bacteriocin c ng dng trong 2 lnh vc
ln: cng ngh dc phm v cng ngh thc phm [59].
a. Trong cng ngh dc phm
Bacteriocin c nghin cu ng dng trong cng ngh dc phm theo
2 hng chnh: probiotic [27] v cht thay th khng sinh [59].
- Probiotic
Probiotic c nh ngha l cc vi sinh vt sng khi c s dng vi
lng ln s em li tc ng c li cho sc khe ca vt ch [55]. Tc
dng ca cc ch phm probiotic c th l trc tip hoc gin tip thng qua
s iu chnh khu h vi sinh vt ng rut [9]. Bacteriocin c cho l ng
vai tr quan trng trong hot ng ca cc probiotic, l mt trong cc tiu ch
la chn chng vi sinh vt lm probiotic [27].
-
14
c nhiu gi thit c a ra v c ch bacteriocin tham gia vo
hot ng ca probiotic. Mt l, bacteriocin gip probiotic xm nhp v
chim u th trong h vi sinh vt ng rut. Hai l, bacteriocin th hin tc
dng khng khun, trc tip c ch cc vi khun gy bnh. Ba l, bacteriocin
mang tn hiu kch thch h min dch ca vt ch. Hin nay, cc nh khoa
hc vn ang tm kim nhng bng chng khng nh chnh xc vai tr v
c ch tc dng ca bacteriocin trong hot ng ca probiotic, nht l cc
bng chng in vivo [27].
- Cht thay th khng sinh
S xut hin v gia tng cc chng vi khun gy bnh a khng thuc l
mt vn ln ca y hc hin nay. gii quyt vn ny, bn cnh vic
s dng hp l cc khng sinh hin c, mt yu cu cp thit c t ra l
nghin cu pht trin cc phng php iu tr mi thay th cho s dng
khng sinh. Bacteriocin l mt trong nhng gii php c xut v
nghin cu [59].
V mt l thuyt, bacteriocin c sinh ra trong t nhin rt a dng nn
kh nng tm ra nhng cht c tc dng khng li cc vi khun gy bnh l rt
ln. Mt khc, do bacteriocin thng c tc dng c hiu trn mt s loi vi
khun nht nh, chng c th c s dng nh nhng thuc thit k ring
cho tng tc nhn gy bnh, nh gim tn sut xut hin vi khun khng
thuc [59].
Cc nghin cu thc nghim cho nhng kt qu bc u kh quan.
y ch nu ra mt vi v d: Nisin, mersacidin, mutacin 1140, lacticin
3147 c bo co v tc dng trn cc vi khun gy bnh khng thuc
nh MRSA, VRE [52]. Nisin F tim vo chut nhim Staphylococcus aureus
cho thy c tc dng kim sot s pht trin ca S. aureus [14]. Lacticin 3147
th hin tc dng c ch Clostridium difficile trn m hnh ex vivo ca rut
-
15
kt [57]. Mt liu duy nht mutacin B-Ny266 tim mng bng vo chut
nhim S. aureus nhy cm vi methicillin cho t l t vong l 0%, trong khi t
l t vong nhm chut i chng (khng dng mutacin B-Ny266) l t 70
n 100% [47].
Mc d vn cn thm nhiu nghin cu c th a bacteriocin vo
ng dng trn thc tin lm sng nhng nhng kt qu trn cho thy tim
nng to ln ca bacteriocin trong vic iu tr cc bnh nhim khun [27].
Trong tng lai, s dng bacteriocin ha hn s l mt liu php an ton v
hiu qu thay th cc khng sinh truyn thng [59].
b. Trong cng ngh thc phm
Bacteriocin ca LAB c nghin cu ng dng lm cht bo qun
trong cng ngh thc phm bi c nhiu u im: c ch hiu qu nhiu vi
khun gy hng v gy bnh trong thc phm (in hnh l Listeria
monocytogenes) [34], thn thin vi mi trng v sc khe con ngi [54].
Chng c s dng di 2 dng: trc tip hoc gin tip [20]. Dng trc tip
l cc bacteriocin c tinh ch, hin nay mi ch c nisin v pediocin PA-
1 c dng rng ri lm cht bo qun thc phm vi cc tn thng mi
nh Nisaplin, ALTA 2341 [20] [52] [54] Dng gin tip l cc LAB c
dng lm ging khi ng (starter culture) hoc b sung vo cc thc phm
ln men, bacteriocin c chng sinh ra s pht huy tc dng khng khun,
gip tng tui th v tnh an ton cho sn phm [20]. tng hiu qu khng
khun, bacteriocin c th c s dng kt hp vi cc phng php khc
nh to mi trng acid, b sung NaCl[34]
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16
Chng 2. NGUYN LIU, THIT B, NI DUNG V PHNG
PHP NGHIN CU
2.1. Nguyn vt liu, thit b
2.1.1. Chng vi khun
a. Vi khun th nghim
Chng vi khun Lactobacillus acidophilus ATCC 4653
b. Vi khun kim nh (ch th)
- Chng vi khun Bacillus subtilis ATCC 6633
- Chng vi khun Escherichia coli ATCC 25922
2.1.2. Ha cht
Bng 2.1: Cc ha cht s dng trong ti
Ha cht Xut x Ha cht Xut x
Acid lactic Trung Quc MgSO4.7H2O Trung Quc
Amoni citrat Trung Quc MnSO4.H2O Trung Quc
Cao nm men c NaCl Trung Quc
Cao tht c NaOH Trung Quc
Ct thy tinh Trung Quc Natri acetat Trung Quc
Ch th mu Trung Quc (NH4)2SO4 Trung Quc
Glucose Trung Quc Pepton n
HCl 1N Trung Quc Thch Vit Nam
K2HPO4 Trung Quc Xanh methylen Trung Quc
KH2PO4 Trung Quc
2.1.3. Mi trng
a. Cng thc mi trng MRS lng
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17
Pepton : 1,0 g Amoni citrat : 0,2 g
Cao tht : 1,0 g Natri acetat : 0,5 g
Cao nm men : 0,5 g MgSO4.7H2O : 0,02 g
Glucose : 2,0 g MnSO4.H2O : 0,004 g
K2HPO4 : 0,2 g Nc my va : 100 ml
b. Cng thc mi trng thch thng
Pepton : 1,0 g
Cao tht : 0,5 g
NaCl : 0,5 g
Thch : 2,3 g
Nc my va : 100 ml
2.1.4. My mc, dng c
a. My mc
Bng 2.2: Cc my mc s dng trong ti
Tn my Xut x Tn my Xut x
Bp cch thy Trung Quc My li tm c (Rotofix)
Cn k thut c (Sartorius) My li tm cao
tc
c (Universal)
Cn phn tch c (Sartorius) Ni hp tit trng Nht (ALP)
L vi sng Hn Quc (Daewoo) T m c (Memmert)
My o vng v
khun
Ty Ban Nha
(IUL)
T m CO2 Nht (Sanyo)
My khuy t Hn Quc (Wisd) T cy v trng Nht (Sanyo)
My lc Vortex c (IKA) T lnh Hn Quc (LG)
Knh hin vi M (Labomed) T sy c (Memmert)
b. Dng c
-
18
Bnh nh mc 50, 100, 1000 ml ng nghim
Bnh nn 100, 250, 500, 1000 ml Pipet chia vch
Chy ci s Pipet Pasteur
Cc c m Pipet tip (u cn)
n cn Pipetman (Pipet Eppendorf)
a petri ng knh 9 cm Que cy u trn
ng ong Tup Eppendorf 1,5 ml
2.2. Ni dung nghin cu
2.2.1. S b xc nh bacteriocin trong dch ln men v sinh khi vi khun
L. acidophilus ATCC 4653
- Xc nh bacteriocin trong dch ln men.
- Xc nh bacteriocin trong sinh khi.
2.2.2. Nghin cu mt s tnh cht ca bacteriocin sinh ra bi vi khun L.
acidophilus ATCC 4653
- nh hng ca pH n hot tnh bacteriocin.
- nh hng ca nhit n hot tnh bacteriocin.
- Kh nng chit bacteriocin bng phng php kt ta (NH4)2SO4.
2.3. Phng php nghin cu
2.3.1. Phng php bo qun ging vi sinh vt
Cc chng vi khun s dng trong ti, bao gm chng vi khun th
nghim L. acidophilus, cc chng vi khun kim nh B. subtilis v E. coli
c bo qun bng phng php cy truyn nh k ln mi trng mi. Cc
thao tc cy truyn c thc hin trong t cy vi sinh. T cy c tit trng
trc khi s dng bng cch lau sch bng cn 70o v bt n UV trong 20
pht.
a. Phng php cy truyn L. acidophilus t mi trng dch th
-
19
Mi trng MRS lng c pha ch theo cng thc mc 2.1.3 a, phn
phi vo cc ng nghim, mi ng 9 ml mi trng, nt kn v tit trng
trong ni hp 115oC trong 20 pht, ngui. Trn u ng ging lng ca
L. acidophilus bng my lc Vortex c hn dch t bo ng nht. Dng
pipet Eppendorf ht 1 ml hn dch t bo t ng ging cy sang ng mi
trng mi. Nui trong t m CO2 5% 37oC trong 24 gi.
Sau thi gian , loi b cc ng khng t yu cu nu c (vi sinh vt
khng mc, mc km, nghi ng b nhim). Cc ng ging c gi k, bo
qun trong t lnh nhit khong 10oC (ngn mt). Vic cy truyn c
thc hin 1 2 tun/ln.
b. Phng php cy truyn B. subtilis v E. coli t mi trng thch
Mi trng thch thng c pha ch theo cng thc mc 2.1.3 b,
phn phi vo cc ng nghim, mi ng 10 ml mi trng, nt kn v tit
trng trong ni hp 121oC trong 20 pht. Sau khi tit trng, t cc ng mi
trng nm nghing khong 15 20o trn mt bn sch thch ng li.
Dng que cy v trng ly mt vng vi sinh vt t ng ging c cy sang
ng mi trng mi, lt que cy trn b mt thch theo hnh zigzag. C B.
subtilis v E. coli u c nui trong t m 37oC trong 24 gi.
Sau thi gian , loi b cc ng khng t yu cu nu c (vi sinh vt
khng mc, mc km, nghi ng b nhim). Cc ng ging c gi k, bo
qun trong t lnh nhit khong 10oC (ngn mt). Vic cy truyn c
thc hin 1 2 thng/ln.
2.3.2. Phng php nhn ging L. acidophilus
Phn phi mi trng MRS lng vo cc bnh nn, mi bnh 100 ml mi
trng, nt kn v tit trng trong ni hp 115oC trong 20 pht, ngui.
Trong t cy v trng, tin hnh cy 1 ng ging lng 10 ml ca L.
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20
acidophilus vo mi bnh nn. Nui trong t m CO2 5% 37oC trong 18 gi
[30].
2.3.3. Phng php x l sinh khi L. acidophilus
Sinh khi L. acidophilus c thu nhn t dch nui cy vi khun sau 18
gi bng cch li tm (4.000 vng/pht, 20 pht [10]). Tch ring phn dch
ni, thu c sinh khi y ng li tm. X l sinh khi thu c theo cc
bc sau:
- Ra sinh khi bng nc ct (1 ln).
- Trn sinh khi t vi ct thy tinh hp tit trng theo t l 1:1,
nghin hn hp to thnh bng chy ci s ph v t bo vi khun.
- B sung NaCl 0,9% vo hn hp thu c theo t l 1:1 (1 ml NaCl
0,9% cho 1 g hn hp), khuy u to thnh hn dch.
- Li tm hn dch thu c bng my li tm cao tc 18.000 vng/pht
trong 20 pht thu dch ni (dch ph t bo).
Kim tra mc ph v t bo ca phng php nghin bng cch lm
tiu bn sinh khi trc v sau khi x l, nhum n vi thuc nhum xanh
methylen ri soi qua knh hin vi vi vt knh du (100x).
2.3.4. Phng php nh gi hot tnh bacteriocin theo c ch khuch tn
qua ging thch
Nguyn tc:
Bacteriocin t mu th trong cc ging khuch tn vo mi trng thch
cy vi sinh vt kim nh v c ch s pht trin ca cc vi sinh vt nhy cm
to ra vng c ch [65]. o ng knh vng c ch to thnh nu c v nh
gi.
Tin hnh:
-
21
- Lm hn dch vi khun kim nh bng cch thm 5 ml nc ct v
trng vo ng ging c ca vi khun kim nh, g nh thnh ng
nghim vo lng bn tay vi khun trn thch phn tn vo nc.
- Mi trng thch thng c pha ch theo cng thc mc 2.1.3 b,
phn phi vo cc bnh nn, nt kn v tit trng trong ni hp 121oC
trong 20 pht.
- Khi mi trng thch thng cn m (khong 45 50oC), hn dch
vi khun kim nh vo bnh nn ng mi trng theo t l 5 ml hn
dch : 100 ml mi trng, lc trn nh vi khun phn tn u trong
mi trng ri ra cc a petri ng knh 9 cm v trng (16 ml mi
trng/a), ngui cho thch ng li.
- Khi thch ng, dng thanh kim loi v trng (ng knh 7,8mm)
khoan to cc ging trn mi trng thch trong a petri.
- Dng pipet Eppendorf nh 50 l mu th vo mi ging thch.
- Cc a petri nh mu th c trong t m 37oC trong 24 gi
cho vi khun kim nh pht trin.
- Sau thi gian , o ng knh vng c ch (nu c) bng my o
ng knh vng v khun v nh gi.
Mt th nghim c tin hnh vi 3 a th song song, ng knh
vng c ch ca mi mu th l kt qu trung bnh o c t 3 a th. Theo
Sifour v cng s, vng c ch nh nht c ngha l 1 mm [62]. Do ng
knh ging thch l 7,8mm, ng knh vng c ch ti thiu c xc nh l
8,8mm.
2.3.5. Phng php kho st nh hng ca pH n hot tnh bacteriocin
Ly 10 ml dch bacteriocin cho mi mu th, xc nh pH bng ch th
mu, t chnh pH v cc gi tr pH nghin cu bng NaOH 1N hoc HCl
1N tit trng [44]. 2 gi [1] nhit phng [62]. nh gi hot tnh
-
22
ca dch bacteriocin chnh pH theo phng php nu mc 2.3.4, so
snh gia cc mu v rt ra nhn xt.
2.3.6.Phng php kho st nh hng ca nhit n hot tnh
bacteriocin
Ly 10 ml dch bacteriocin cho mi mu th, un trn bp cch thy
cc gi tr nhit nghin cu trong 30 pht [44]. ngui nhit phng.
nh gi hot tnh ca dch bacteriocin x l nhit theo phng php
nu mc 2.3.4, so snh vi mu chun i chiu l dch bacteriocin khng
x l nhit v rt ra nhn xt.
2.3.7. Phng php kho st kh nng chit bacteriocin bng (NH4)2SO4
Nguyn tc:
Bacteriocin c bn cht protein nn to kt ta khi nng (NH4)2SO4
ln [56]. Tch ring phn ta bacteriocin v phn dch, nh gi hot tnh
bacteriocin ca mi phn. Bacteriocin kt ta cng nhiu th hot tnh ca
phn ta cng ln, hot tnh ca phn dch cng nh.
Tin hnh:
- Lm lnh dch bacteriocin bng cch trong t lnh 4oC [1] trong
khong 1 gi.
- Thm dn (NH4)2SO4 theo cc nng mui nghin cu, c kt hp
khuy t [1].
- Li tm dch bacteriocin ha tan mui bng my li tm cao tc
10.000 vng/pht trong 20 pht phn ring phn ta v phn dch
[56].
- Ha tan phn ta trong m phosphat pH 7,0 [56] theo t l khong 1
ml m cho phn ta ca 30 ml dch bacteriocin ban u [46].
- nh gi hot tnh bacteriocin ca phn ta v phn dch theo phng
php nu mc 2.3.4, so snh gia cc mu v rt ra nhn xt.
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23
Chng 3. THC NGHIM, KT QU V BN LUN
3.1. S b xc nh bacteriocin trong dch ln men v sinh khi vi khun
L. acidophilus ATCC 4653
L. acidophilus l vi khun Gram (+), do v mt l thuyt bacteriocin
ca n c th tn ti di 2 dng: dng gn vi t bo v dng ngoi bo [66].
V vy, ni dung u tin ca ti l tin hnh xc nh s b bacteriocin
trong dch ln men v sinh khi ca chng L. acidophilus th nghim.
Mc ch:
- Xc nh v tr tn ti ca bacteriocin sinh ra bi vi khun L.
acidophilus ATCC 4653.
- Nhn nh s b v hot tnh ca bacteriocin sinh ra bi L. acidophilus
ATCC 4653 trn cc vi khun kim nh c s dng.
Cch tin hnh:
Nui cy L. acidophilus ATCC 4653 theo mc 2.3.2. Li tm dch nui
cy 4.000 vng/pht trong 20 pht tch ring phn dch ni (dch ln
men) v sinh khi [10]. X l sinh khi thu c theo mc 2.3.3 thu dch
ph t bo. Tin hnh xc nh bacteriocin trong dch ln men v dch ph t
bo theo phng php nu mc 2.3.4; trong mu trng i vi dch
ln men l dung dch acid lactic c pH 4 (tng ng pH dch ln men), i
vi dch ph t bo l dung dch NaCl 0,9%. Cc vi khun kim nh c la
chn l B. subtilis ATCC 6633, i din cho vi khun Gram (+) v E. coli
ATCC 25922, i din cho vi khun Gram (-) [44]. Th nghim c lp li 3
ln trn 3 m nui cy c lp ca L. acidophilus ATCC 4653.
Kt qu:
3.1.1. Xc nh bacteriocin trong dch ln men
-
24
Kt qu xc nh bacteriocin trong dch ln men ca L. acidophilus
ATCC 4653 c th hin trong bng 3.1.
Bng 3.1: Kt qu xc nh bacteriocin trong dch ln men ca L. acidophilus
ATCC 4653
Ln th
nghim
ng knh vng c ch (mm)
Trn B. subtilis ATCC 6633 Trn E. coli ATCC 25922
Dch ln men Acid lactic
pH 4 Dch ln men
Acid lactic
pH 4
Ln 1 16,0 (-) 18,7 (-)
Ln 2 16,2 (-) 19,8 (-)
Ln 3 17,7 (-) 22,5 (-)
Trung bnh 16,6 20,3
Ghi ch: (-): khng xut hin vng c ch
Nhn xt v bn lun:
Kt qu bng 3.1 cho thy: trong c 3 ln th nghim, dch ln men ca
L. acidophilus ATCC 4653 u to c vng c ch trn cc chng vi khun
kim nh B. subtilis ATCC 6633 v E. coli ATCC 25922. Mu trng l dung
dch acid lactic c pH 4 (tng ng pH dch ln men) khng to vng c
ch trong tt c cc th nghim tng ng, iu ny cho php loi tr tc dng
ca acid hu c c trong dch ln men. T , c th rt ra 2 kt lun sau:
Mt l, bacteriocin c trong dch ln men ca L. acidophilus ATCC
4653. Kt qu ny ph hp vi nhiu nghin cu c v bacteriocin ca L.
acidophilus [24] [39] [49] [67] [72]. Trong cc nghin cu ny, cc tc gi
u s dng dch ln men ca vi khun thu nhn bacteriocin.
-
25
Hai l, bacteriocin trong dch ln men ca L. acidophilus ATCC 4653 c
tc dng c ch i vi c B. subtilis v E. coli, tc l th hin hot tnh trn
c vi khun Gram (+) v Gram (-). y l mt c im khng thng gp
bacteriocin ca LAB. Theo Parada v cng s, bacteriocin ca LAB thng
c tc dng trn vi khun Gram (+) nhng t tc dng trn vi khun Gram (-)
[54]. Nm 2010, Ahmed v cng s tin hnh thng k 16 bacteriocin
c tm thy L. acidophilus (Ph lc 5), trong 13 bacteriocin c ph tc
dng gii hn trn vi khun Gram (+). Trong 3 bacteriocin cn li c tc dng
c ch vi khun Gram (-), ng ch nht l acidocin AA11 vi ph tc dng
trn c B. subtilis v E. coli, ging vi bacteriocin ca L. acidophilus ATCC
4653 [11].
Tin hnh so snh vng c ch to bi bacteriocin trong dch ln men
ca L. acidophilus ATCC 4653 trn B. subtilis v trn E. coli cho thy: V
mt nh tnh, vng c ch ca bacteriocin trn B. subtilis trong v r hn so
vi trn E. coli (Hnh 3.1 v 3.2). V mt nh lng, trong c 3 ln th
nghim, ng knh vng c ch khi dng E. coli lm vi khun kim nh u
ln hn so vi khi dng B. subtilis. C th, ng knh vng c ch trn E.
coli trong 3 ln th nghim ln lt l 18,7; 19,8 v 22,5mm; trong khi ng
knh vng c ch trn B. subtilis tng ng ch l 16,0; 16,2 v 17,7mm.
ng knh vng c ch trung bnh ca 3 ln th nghim trn E. coli l
20,3mm, ln hn 3,7mm so vi gi tr tng ng trn B. subtilis (16,6mm).
Trong mt nghin cu ca Abo-Amer nm 2007 v acidocin AA11 t L.
acidophilus AA11, tc gi khng cng b s liu c th v ng knh vng
c ch to bi bacteriocin trn cc vi khun kim nh m ch cho bit ng
knh vng c ch trn B. subtilis v E. coli u ln hn 10 mm [10]. Kt qu
thc nghim thu c trong ti cng ph hp vi cng b ny.
-
26
Hnh 3.1: (1) Vng c ch to bi
dch ln men ca L. acidophilus
ATCC 4653 trn vi khun kim nh
B. subtilis ATCC 6633
Hnh 3.2: (1) Vng c ch to bi
dch ln men ca L. acidophilus
ATCC 4653 trn vi khun kim nh
E. coli ATCC 25922
3.1.2. Xc nh bacteriocin trong sinh khi
Kt qu xc nh bacteriocin trong sinh khi ca L. acidophilus ATCC
4653 c th hin trong bng 3.2.
Bng 3.2: Kt qu xc nh bacteriocin trong sinh khi ca L. acidophilus
ATCC 4653
Ln th
nghim
ng knh vng c ch (mm)
Trn B. subtilis ATCC 6633 Trn E. coli ATCC 25922
Dch ph t
bo NaCl 0,9%
Dch ph t
bo NaCl 0,9%
Ln 1 (-) (-) (-) (-)
Ln 2 (-) (-) (-) (-)
Ln 3 (-) (-) (-) (-)
Ghi ch: (-): khng xut hin vng c ch
1 1
-
27
Nhn xt v bn lun:
Kt qu bng 3.2 cho thy: trong c 3 ln th nghim, dch ph t bo
ca L. acidophilus ATCC 4653 u khng to vng c ch trn cc chng vi
khun kim nh B. subtilis ATCC 6633 v E. coli ATCC 25922. Mt khc,
theo kt qu mc 3.1.1, bacteriocin ca L. acidophilus ATCC 4653 c hot
tnh trn 2 chng vi khun kim nh ny. V vy, c th s b kt lun
bacteriocin khng c trong dch ni bo ca L. acidophilus ATCC 4653.
Theo Tagg v cng s, c im mi trng l yu t quyt nh t l cc
dng tn ti ca bacteriocin vi khun Gram (+) [66]. Trong ti ny, L.
acidophilus ATCC 4653 c nui cy trong mi trng MRS lng 37oC
trong 18 gi v khng kim sot pH mi trng trong qu trnh nui cy.
Khng loi tr kh nng bacteriocin ca L. acidophilus ATCC 4653 c th
tn ti dng ni bo nu thay i iu kin nui cy vi khun.
Phng php x l sinh khi c thc hin trong ti l nghin hn
hp sinh khi t ca L. acidophilus trn vi ct thy tinh bng chy ci s
(mc 2.3.3). y l mt phng php ph t bo thch hp vi qui m phng
th nghim [4]. u im ca phng php ny l n gin, d thc hin,
khng yu cu ha cht, thit b t tin, khng c nguy c gy nh hng ti
hot tnh bacteriocin nh mt s phng php khc. Tuy nhin, vi phng
php nghin, t bo vi khun mi c ph v v mt c hc. Khng loi tr
kh nng phng php ny c th cha thch hp gii phng bacteriocin t
sinh khi.
Trong phm vi ti ny, do cha c iu kin th nghim cc iu kin
nui cy v cc phng php x l sinh khi khc, vic xc nh bacteriocin
trong sinh khi ca L. acidophilus ATCC 4653 tm dng li vic kt lun
bacteriocin khng c trong dch ni bo ca vi khun.
-
28
Tm li, kt qu mc 3.1 cho thy, trong cc iu kin thc nghim ca
ti, bacteriocin ca L. acidophilus ATCC 4653 khng tn ti dng ni
bo m hon ton dng ngoi bo. V vy, dch ln men ca L. acidophilus
ATCC 4653 c coi l dch bacteriocin th, c s dng thc hin cc
th nghim tip theo nghin cu v tnh cht ca bacteriocin. Bacteriocin ny
c hot tnh trn c vi khun Gram (+) (B. subtilis ATCC 6633) v vi khun
Gram (-) (E. coli ATCC 25922), trong vng c ch to bi bacteriocin trn
B. subtilis c ng knh nh hn nhng li trong v r hn so vi trn E.
coli. Do , thun tin cho vic quan st v n gin ha qu trnh th
nghim, B. subtilis c la chn lm vi khun kim nh nh gi hot
tnh bacteriocin trong cc th nghim tip theo.
3.2. Nghin cu mt s tnh cht ca bacteriocin sinh ra bi vi khun L.
acidophilus ATCC 4653
Cc bacteriocin khc nhau c th c nhng c im ring rt a dng
[32], dn n phng php sn xut v phm vi ng dng khc nhau. V vy,
ni dung tip theo ca ti l tin hnh nghin cu mt s tnh cht c bn
ca bacteriocin sinh ra bi chng L. acidophilus th nghim, bao gm nh
hng ca pH, nhit n hot tnh bacteriocin v kh nng chit
bacteriocin bng phng php kt ta (NH4)2SO4.
3.2.1. Nghin cu nh hng ca pH n hot tnh bacteriocin ca L.
acidophilus ATCC 4653
nh hng ca pH l mt ni dung quan trng khi nghin cu v n
nh ca bacteriocin [54] [66]. Khong pH hot ng ca cc bacteriocin khc
nhau c th rt khc nhau, c th rt hp nh boticin P (khong pH hot
ng 6,5 7,5), c th rt rng nh butyricin 7423 (khong pH hot ng 2
12) [66]. Xc nh khong pH hot ng ca bacteriocin cho php s dng
-
29
bacteriocin c hiu qu. Do , bacteriocin ca chng L. acidophilus th
nghim c nghin cu v nh hng ca pH n hot tnh bacteriocin.
Mc ch:
Xc nh s b khong pH hot ng ca bacteriocin sinh ra bi L.
acidophilus ATCC 4653.
Cch tin hnh:
Nui cy L. acidophilus ATCC 4653 theo mc 2.3.2. Li tm dch nui
cy 4.000 vng/pht trong 20 pht loi sinh khi, thu dch ni l dch
bacteriocin th (dch ln men) [10]. Kho st nh hng ca pH n hot tnh
bacteriocin theo phng php nu mc 2.3.5, trong cc gi tr pH
c nghin cu l 2, 4, 6, 8. Vi khun kim nh c s dng l B. subtilis
ATCC 6633. Th nghim c lp li 3 ln trn 3 m nui cy c lp ca L.
acidophilus ATCC 4653.
Kt qu:
Kt qu nh gi hot tnh bacteriocin ca L. acidophilus ATCC 4653
cc gi tr pH nghin cu c th hin trong bng 3.3.
Nhn xt v bn lun:
Kt qu bng 3.3 cho thy trong cc gi tr pH nghin cu, bacteriocin
ca L. acidophilus ATCC 4653 th hin hot tnh tt nht pH 2, vi ng
knh vng c ch trung bnh qua 3 ln th nghim l 18,8mm. pH cng tng
th ng knh vng c ch cng nh, hot tnh bacteriocin cng yu. Hot
tnh bacteriocin gim t ngt pH 6 (gn trung tnh), vi gim ng
knh trung bnh so vi pH 2 l 37,8%, cao hn hn so vi gim 9,0% pH
4. n gi tr pH 8 (mi trng kim nh), ng knh vng c ch trung
bnh ch cn 9,7mm, gim 48,4% so vi pH 2 v ch ln hn 0,9mm so vi
ng knh vng c ch ti thiu c ngha (8,8mm) [62]. iu ny chng t
-
30
bacteriocin ca L. acidophilus ATCC 4653 km hot ng trong mi trng
kim.
Bng 3.3: Hot tnh bacteriocin ca L. acidophilus ATCC 4653 cc gi tr
pH nghin cu trn vi khun kim nh B. subtilis ATCC 6633
Ln th
nghim
ng knh vng c ch (mm)
pH 2 pH 4 pH 6 pH 8
Ln 1 17,6 16,5 11,7 9,3
Ln 2 18,5 16,8 11,2 9,7
Ln 3 20,2 18,1 12,1 10,2
Trung bnh 18,8 17,1 11,7 9,7
(%) 0 9,0 37,8 48,4
Ghi ch: : gim ng knh trung bnh pH x so vi pH 2
(%) =
vi d(2): ng knh vng c ch trung bnh pH 2
d(x): ng knh vng c ch trung bnh pH x
Nh vy, t nhng kt qu trn, c th nhn nh bacteriocin ca L.
acidophilus ATCC 4653 ch yu hot ng pH acid v pH cng nh th
hot tnh bacteriocin cng ln. y l c im ca a s bacteriocin t LAB
m in hnh l nisin [21]. Nisin hu nh ch pht huy tc dng trong mi
trng acid (hot tnh cao nht pH 2, b bt hot pH trn 8) nn c s
dng lm cht bo qun trong cc thc phm c tnh acid [31]. S tng hot
tnh ca bacteriocin khi pH gim c Jack v cng s l gii theo mt s gi
thit nh sau [36]:
- Khi pH gim, bacteriocin t to kt tp hn, do c nhiu phn t
tham gia tn cng tiu dit t bo ch.
-
31
- Khi pH gim, bacteriocin t gn vi t bo hn, a s bacteriocin
dng ngoi bo pht huy tc dng dit khun.
- pH gim to iu kin cho bacteriocin d thm qua vch t bo ch.
- Phn ng tng tc gia bacteriocin vi t bo ch b c ch trong mi
trng pH cao.
Hnh 3.3: nh hng ca pH n hot tnh bacteriocin ca L.
acidophilus ATCC 4653
3.2.2. Nghin cu nh hng ca nhit n hot tnh bacteriocin ca L.
acidophilus ATCC 4653
Cng vi nh hng ca pH, bn nhit cng l mt ni dung quan
trng khi nghin cu v bacteriocin [54], c ngha c v l thuyt v thc
tin. V l thuyt, l mt trong nhng tiu ch c bn trong vic m t v
phn loi bacteriocin ca LAB [53]. V thc tin, n lin quan n kh nng
ng dng ca bacteriocin, nht l trong cng ngh thc phm khi nhiu qui
18.8
17.1
11.7
9.7
0
2
4
6
8
10
12
14
16
18
20
2 4 6 8 pH
ng
kn
h v
ng
c
ch
trung
bn
h (
mm
)
-
32
trnh ch bin thc phm yu cu phi s dng nhit [62]. V vy, bacteriocin
ca chng L. acidophilus th nghim c nghin cu v nh hng ca
nhit n hot tnh bacteriocin.
Mc ch:
nh gi bn nhit ca bacteriocin sinh ra bi L. acidophilus ATCC
4653.
Cch tin hnh:
Thu dch bacteriocin th (dch ln men) tng t mc 3.2.1. Kho st
nh hng ca nhit n hot tnh bacteriocin theo phng php nu
mc 2.3.6, trong cc gi tr nhit c nghin cu l 70oC, 80oC, 90oC
v 100oC. Vi khun kim nh c s dng l B. subtilis ATCC 6633. Th
nghim c lp li 3 ln trn 3 m nui cy c lp ca L. acidophilus
ATCC 4653.
Kt qu:
Kt qu nh gi hot tnh bacteriocin ca L. acidophilus ATCC 4653
sau khi c x l nhit c th hin trong bng 3.4.
Bng 3.4: Hot tnh bacteriocin ca L. acidophilus ATCC 4653 sau khi c
x l nhit trn vi khun kim nh B. subtilis ATCC 6633
Ln th
nghim
ng knh vng c ch (mm)
Khng x
l nhit 70
oC 80
oC 90
oC 100
oC
Ln 1 18,1 18,7 19,4 19,4 19,3
Ln 2 18,7 18,4 18,0 18,3 18,3
Ln 3 16,3 16,7 16,2 16,1 16,3
Trung bnh 17,7 17,9 17,9 17,9 18,0
-
33
Hnh 3.4: nh hng ca nhit n hot tnh bacteriocin ca L.
acidophilus ATCC 4653
Nhn xt v bn lun:
Kt qu bng 3.4 cho thy sau khi x l nhit dch ln men cc gi tr
nhit nghin cu, hot tnh bacteriocin ca L. acidophilus ATCC 4653 hu
nh khng thay i. C th, ng knh vng c ch trung bnh qua 3 ln th
nghim sau khi un dch ln men ca chng L. acidophilus th nghim cc
nhit 70oC, 80oC, 90oC, 100oC trong 30 pht u xp x nhau v xp x vi
ng knh vng c ch trung bnh khi dch ln men khng c x l nhit
(17,7mm). T , c th nhn nh bacteriocin ca L. acidophilus c tnh bn
nhit.
Trong cc bacteriocin ca LAB, tnh bn nhit l c tnh c bn ca lp
I v lp II bao gm cc bacteriocin c cu trc l cc peptid phn t lng
17.7 17.9 17.9 17.9 18.0
0
2
4
6
8
10
12
14
16
18
20
Khng x l nhit
un 70oC /30 pht
un 80oC /30 pht
un 90oC /30 pht
un 100oC /30 pht
ng
kn
h v
ng
c
ch
trung
bn
h (
mm
)
un 70oC/ 30 pht
un 80oC/ 30 pht
un 90oC/ 30 pht
un 100oC/ 30 pht
Khng x l nhit
-
34
nh (mc 1.2.2). Theo De Vuyst v Vandamme, c tnh bn nhit ca cc
bacteriocin ny l do chng c cu to n gin (khng c cu trc bc 3)
hoc c cu trc hnh cu c n nh nh cc lin kt ng ha tr [21].
Oscariz v Pisabarro li nhn mnh vai tr ca cc cu ni sulfit v disulfit
ni phn t trong vic n nh cu trc, m bo tnh bn nhit ca
bacteriocin. Cc tc gi cn a ra gi thuyt rng s cu ni cng tng th
bacteriocin cng bn nhit [53].
Trong s cc bacteriocin c tm ra L. acidophilus, a s c tnh
bn nhit ging vi bacteriocin trong nghin cu, ch c mt s t c ghi
nhn l nhy cm vi nhit gm c bacteriocin ca L. acidophilus AC1 [45]
v acidophilucin A ca L. acidophilus LAPT 1060 [68].
Tm li, ging vi a s bacteriocin c tm ra L. acidophilus,
bacteriocin ca L. acidophilus ATCC 4653 c tnh bn nhit, vi nhit cao
nht c x l l 100oC trong 30 pht.
3.2.3. Nghin cu kh nng chit bacteriocin ca L. acidophilus ATCC
4653 bng phng php kt ta (NH4)2SO4
Kt ta bng (NH4)2SO4 l mt phng php thng c p dng
chit bacteriocin [20]. So vi cc phng php chit bacteriocin khc nh hp
ph - gii hp ph, chit bng dung mi hu c, phng php ny c u
im l n gin, d thc hin, ha cht r tin, khng c hi. Do ,
phng php kt ta bng (NH4)2SO4 c la chn bc u nghin cu
kh nng chit bacteriocin t dch ln men ca chng L. acidophilus th
nghim.
Mc ch:
Nhn nh s b v kh nng chit bacteriocin t dch ln men ca L.
acidophilus ATCC 4653 bng phng php kt ta (NH4)2SO4 theo cc nng
mui khc nhau.
-
35
Cch tin hnh:
Thu dch bacteriocin th (dch ln men) tng t mc 3.2.1. Kho st
kh nng chit bacteriocin bng (NH4)2SO4 theo phng php nu mc
2.3.7, trong cc nng (NH4)2SO4 c nghin cu l 20; 30; 40; 50; 60;
70 v 80% (khi lng/th tch). Vi khun kim nh c s dng l B.
subtilis ATCC 6633.
Kt qu:
Kt qu nh gi hot tnh bacteriocin ca phn ta v phn dch thu
c sau khi b sung (NH4)2SO4 vi cc nng khc nhau vo dch ln men
ca L. acidophilus ATCC 4653 c th hin trong bng 3.5.
Bng 3.5: Hot tnh bacteriocin ca phn ta v phn dch thu c khi b
sung (NH4)2SO4 vo dch ln men ca L. acidophilus ATCC 4653 vi vi
khun kim nh l B. subtilis ATCC 6633
Nng
(NH4)2SO4 (%)
ng knh vng c ch (mm)
Phn ta Phn dch
20 Khng to ta
30 13,2 16,6
40 13,7 15,3
50 13,9 14,6
60 14,7 14,2
70 15,6 13,7
80 Mui khng tan ht, ln vi ta
Nhn xt v bn lun:
Kt qu bng 3.5 cho thy, trong dy nng (NH4)2SO4 nghin cu (t
20 n 80%), nng mui thp nht cho php tch c ta bacteriocin t
-
36
dch ln men ca L. acidophilus ATCC 4653 l 30% v cao nht l 70%.
nng (NH4)2SO4 20%, bacteriocin cha to kt ta; cn nng
(NH4)2SO4 80%, mui khng tan ht v ln vi phn ta bacteriocin.
Trong khong nng (NH4)2SO4 tch c ta bacteriocin (t 30% n
70%), nhn thy: khi tng nng (NH4)2SO4, hot tnh bacteriocin ca phn
ta tng dn, trong khi hot tnh bacteriocin ca phn dch gim dn. ng
knh vng c ch ca phn ta nng (NH4)2SO4 70% l 15,6mm, tng
2,4mm so vi nng 30% (13,2mm). Ngc li, ng knh vng c ch
ca phn dch nng (NH4)2SO4 70% l 13,7mm, gim 2,9mm so vi
nng 30% (16,6mm). T , c th kt lun lng bacteriocin kt ta tng
dn theo lng (NH4)2SO4 s dng.
Hnh 3.5: S thay i hot tnh bacteriocin ca phn ta v phn dch theo
nng (NH4)2SO4 s dng
0
2
4
6
8
10
12
14
16
18
30 40 50 60 70
Phn ta
Phn dch
Nng (NH4)2SO4 (%)
ng
kn
h v
ng
c
ch
(mm
)
-
37
Trong ti ny, nng (NH4)2SO4 cao nht trc khi mui bo ha
c nghin cu l 70%. nng ny phn dch vn cn hot tnh
bacteriocin (to vng c ch c ng knh 13,7mm), chng t bacteriocin
ca L. acidophilus ATCC 4653 vn cha c chit ht. Cng nng ny,
ng knh vng c ch to bi phn ta l 15,6mm, ln hn 1,9mm so vi
vng c ch ca phn dch (13,7mm). Mt khc, phn ta c c c 30
ln so vi phn dch (mc 2.3.7). T , c th d on hiu sut chit
bacteriocin t dch ln men ca L. acidophilus ATCC 4653 bng phng
php kt ta (NH4)2SO4 khng cao.
Phng php kt ta bng (NH4)2SO4 c nhiu tc gi s dng
chit bacteriocin t LAB, hiu sut chit t c c th rt cao ln ti 95%
[46] nhng cng c khi kh thp ch gn 20% [12]. Trong ti ny, hiu sut
chit bacteriocin bng phng php kt ta (NH4)2SO4 cha c tnh ton c
th m mi ch a ra nhng nhn nh bc u. la chn phng php
chit bacteriocin thch hp cn tin hnh thm nhng kho st v nh gi
trn mt s phng php chit khc. Thc t c nhng nghin cu la
chn chit bacteriocin bng cc phng php khc nh chit bng ethanol [1],
butanol [15].
Tm li, t cc kt qu mc 3.2, c th tm lc mt s tnh cht c
bn ca bacteriocin sinh ra bi L. acidophilus ATCC 4653 nh sau:
V khong pH hot ng: bacteriocin ch yu hot ng pH acid, pH
cng nh th hot tnh bacteriocin cng ln.
V bn nhit: bacteriocin c tnh bn nhit, hot tnh bacteriocin hu
nh khng thay i sau khi x l nhit 100oC trong thi gian 30 pht.
V kh nng chit bacteriocin bng phng php kt ta (NH4)2SO4:
bacteriocin c th c chit t dch ln men vi nng (NH4)2SO4 t 30%
-
38
n 70%, lng bacteriocin kt ta tng dn theo lng (NH4)2SO4 s dng.
nng (NH4)2SO4 70%, bacteriocin vn cha ta ht.
Trn y mi l nhng m t mang tnh s b v bacteriocin ca L.
acidophilus ATCC 4653. m t chi tit bacteriocin ny cn c nhng
nghin cu su hn.
-
39
KT LUN V KIN NGH
1. Kt lun:
ti hon thnh c cc mc tiu ra v thu c mt s kt qu
nh sau:
- Trong cc iu kin thc nghim ca ti, bacteriocin ca chng L.
acidophilus ATCC 4653 c tm thy c trong dch ln men v khng
c trong sinh khi. Bacteriocin ny th hin tc dng c ch trn c vi
khun Gram (+) (B. subtilis ATCC 6633) v vi khun Gram (-) (E. coli
ATCC 25922). Trn B. subtilis, bacteriocin to vng c ch trong, r
hn v c ng knh nh hn so vi trn E. coli. ng knh vng c
ch trung bnh ca 3 ln th nghim trn B. subtilis l 16,6mm, cn trn
E. coli l 20,3mm.
- Nhn nh s b mt s tnh cht ca bacteriocin sinh ra bi chng L.
acidophilus ATCC 4653:
Bacteriocin a pH acid, hot tnh tng khi pH gim. Trong cc
gi tr pH nghin cu, bacteriocin th hin hot tnh cao nht
pH 2 vi ng knh vng c ch trung bnh qua 3 ln th
nghim l 18,8mm.
Bacteriocin c tnh bn nhit. Sau khi un 100oC trong 30 pht,
hot tnh bacteriocin hu nh khng thay i.
Bacteriocin c th c chit t dch ln men bng phng php
kt ta (NH4)2SO4 vi nng mui t 30% n 70% (khi
lng/th tch), lng bacteriocin kt ta tng dn khi tng lng
(NH4)2SO4 s dng. nng (NH4)2SO4 70%, phn ta
bacteriocin to vng c ch c ng knh 15,6mm; phn dch
sau khi loi ta bacteriocin vn to vng c ch c ng knh
-
40
13,7mm, cho thy bacteriocin trong dch ln men vn cha kt
ta ht.
2. Kin ngh:
ti c th c tip tc pht trin theo mt s hng nh sau:
- Kho st nh hng ca iu kin nui cy n kh nng sinh
bacteriocin ca L. acidophilus ATCC 4653 theo cc thng s nh nhit
, pH, thi gian nui cy; t la chn c iu kin ti u sao
cho thu c nhiu bacteriocin nht.
- Tip tc nghin cu cc phng php chit xut v tinh ch bacteriocin
ca L. acidophilus ATCC 4653, t xy dng qui trnh chit tch ti
u v nh danh bacteriocin nghin cu.
-
TI LIU THAM KHO
Ting Vit
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cht ca bacteriocin c tng hp bi vi khun lactic phn lp t sa b
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6. Trn Ngc Tn, inh Th Thu Hng, Th Huyn, Trng Nam Hi
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55.
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Linh, Th Huyn, L Vn Trng, Trng Nam Hi (2008), Tng hp v
biu hin gen m ha enterocin vng AS-48 ca vi khun Enterococcus
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faecalis trong t bo Escherichia coli, Tp ch Cng ngh Sinh hc, 6(3),
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hc ca chng vi khun lactic sinh tng hp bacteriocin, Tp ch Khoa hc
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Ting Anh
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-
PH LC
Ph lc 1: Phn loi s b cc bacteriocin v c im ca mt s bacteriocin tiu biu cho mi nhm [26]
(A)
Protein-
bacteriocin
Lp Phn lp
Tn Khi lng phn t (kDa)
C ch tc dng
Vi khun Gram (-)
Escherichia coli Colicin A A, E1 E9, K, L, N, S4,
U, Y
40 n 80 Nuclease / To l
B B, D, H, Ia,
Ib, M, 5, 10
40 n 80 Nuclease / To l
Pseudomonas
aeruginosa
Pyocin Typ R Pyocin R2 270 aa To l
Typ S Pyocin S1,
S2, AP41
75/84/
94
Ging phage
Typ F Pyocin F Ging phage
Hafnia alvei Alveicin Ging colicin
Alveicin A, B 408/358 aa To l
Klebsiella
pneumonia
Klebicin Ging colicin
Klebicin C, D 96 Nuclease
Serratia
plymithicum
Serracin Serracin P 66 Ging phage
Xanthomonas
campestris
Glynericin Glynericin A 50 Ging phage
Yersinia
enterocolitica
Enterocoliticin 669 Ging phage
Erwinia
carotovora
Carotovoricin Carotovoricin
Er
68/76 Ging phage
Vi khun Gram (+)
Lactobacillus
helveticus
Helveticin J III 37,5 Cha r
Streptococcus
milleri
Millericin III 30 Thy phn peptidoglycan
Enterococcus
faecalis
Enterolysin III 34,5 Thy phn peptidoglycan
Staphylococcus
aureus
Lysostaphin III 25 Thy phn peptidoglycan
-
(B)
Peptid-
bacteriocin
Lp Phn lp
Tn Khi lng phn t (kDa)
C ch tc dng
Vi khun Gram (-)
Escherichia coli Microcin I Microcin
B17
3,1 Enzym ni bo
II IIa Microcin V 8,8 To l
IIb Microcin
E492
7,9 To l
Vi khun Gram (+)
I
(Hay
lantibiotic)
Typ A A1 Nisin 3,5 To l
A2 Lacticin 481 3 To l
Typ B Mersacidin 2
II IIa Pediocin 4,6 To l
IIb Plantaricin
E/F
3,5/3,7 To l
IIc Carnocyclin
A
5,8 To l
IId Lactococcin
A
5,8 To l
Cyanobacteria
Prochloron
didemni
Ging microcin
Patellamides 0,7
Ghi ch: aa: acid amin
-
Ph lc 2: Mt s bacteriocin ca LAB v nhng tnh cht c bn [55]
Loi sn xut
Bacteriocin Ph tc dng c im
Lactococcus
lactis subsp.
lactis
Lactococcus
lactis subsp.
cremoris
Nisin Vi khun Gram (+) Lantibiotic, 3,5 kDa, 34 acid amin, c sn
phm thng mi
Lacticin 3147 Clostridium sp.
Listeria monocytogenes
Staphylococcus aureus
Streptococcus
dysgalactiae
Enterococcus faecalis
Propionibacterium acne
Streptococcus mutans
Lantibiotic, 4,2 kDa, bn nhit, th hin hot tnh
pH acid v sinh l
Lactococcin B Lactobacillus Bacteriocin lp II, xp x 5 kDa, ph tc dng hp
Lactobacillus
acidophilus
Acidocin CH5 Vi khun Gram (+) Bacteriocin lp II, to kt tp khi lng ln
Lactacin F Lactobacillus fermentum
Enterococcus faecalis
Lactobacillus delbrueckii
Lactobacillus helveticus
Bacteriocin lp II, 6,3 kDa, 57 acid amin, bn
nhit
Lactacin B Lactobacillus delbrueckii
Lactabacillus helveticus
Lactobacillus bulgaricus
Lactococcus lactis
Bacteriocin lp II, 6,3 kDa, bn nhit, ch c
pht hin khi pH mi trng nui cy c gi
n nh t 5,0 6,0
Lactobacillus
amylovorus
Lactobin A Lactobacillus acidophilus
Lactobacillus delbrueckii
Bacteriocin lp II, 4,8 kDa, 50 acid amin, ph
tc dng hp
Lactobacillus
casei
Lactocin 705 Listeria monocytogenes
Lactobacillus plantarum
Bacteriocin lp II, gm 2 peptid; mi peptid gm
33 acid amin, khi lng phn t 3,4 kDa
Leuconostoc
gelidum
Leucocin A Lactobacillus
Enterococcus faecalis
Listeria monocytogenes
Bacteriocin lp II, 3,9 kDa, 37 acid amin, bn
pH acid, bn nhit
Leuconostoc
mesenteroides
Mesentericin
Y105
Enterococcus faecalis
Listeria monocytogenes
Bacteriocin lp II, 3,8 kDa, 37 acid amin, bn
nhit
Pediococcus
acidilactici
Pediocin F Vi khun Gram (+) Bacteriocin lp II, 4,5 kDa, bn vi nhit v
-
dung mi hu c, hot ng trong khong pH
rng
Pediocin PA-1 Listeria monocytogenes Bacteriocin lp II, 4,6 kDa, 44 acid amin
Pediocin AcH Vi khun Gram (+) Vi khun Gram (-) khi b
sung stress
Bacteriocin lp II, 4,6 kDa, 44 acid amin, ph
tc dng rng
Pediococcus
pentosaceous
Pediocin A Lactobacillus
Lactococcus
Leuconostoc
Pediococcus
Staphylococcus
Enterococcus
Listeria
Clostridium
Bacteriocin lp II, 2,7 kDa, bn nhit
Enterococcus
faecium
Enterocin A Listeria monocytogenes
Pediococcus
Bacteriocin lp II, 4,8 kDa, 47 acid amin, bn
nhit
Lactobacillus
sake
Lactocin S Lactobacillus
Leuconostoc
Pediococcus
Lantibiotic, 3,7 kDa, th hin hot tnh trong khong pH 4.5 7.5
Sakacin P Listeria monocytogenes Bacteriocin lp II, 4,4 kDa, bn nhit
Lactobacillus
curvatus
Curvacin A Listeria monocytogenes
Enterococcus faecalis
Bacteriocin lp II, 4,3 kDa
Lactobacillus
helveticus
Helveticin J Lactobacillus bulgaricus
Lactococcus lactis
Bacteriocin lp III, 37 kDa, ph tc dng hp,
km bn nhit
-
Ph lc 3: Mt s phng php nh gi hot tnh bacteriocin
Phng php Nguyn tc Cch tin hnh
Phng php
spot-on-lawn
(Spot-on-lawn
assay) [57]
Bacteriocin c kh nng khuch
tn trong mi trng c hoc
bn c, do mu th cha
bacteriocin s c ch s pht
trin ca vi sinh vt ch th, to
thnh vng c ch xung quanh
mu th.
Mu th c nh git ln mi
trng c (~2% thch). hn
hp vi sinh vt ch th trn trong
mi trng thch mm (~0.7%
thch) ln trn. qua m, nh
gi vng c ch to thnh.
Phng php
khuch tn qua
khoanh giy lc
(Disc diffusion
assay) [57]
Nh mu th ln cc khoanh giy
lc v trng, kh ri t ln b
mt mi trng c cy vi sinh
vt ch th. qua m, nh gi
vng c ch to thnh.
Phng php
khuch tn qua
ging thch
(Well diffusion
assay) [57]
Trn mi trng c cy vi
sinh vt ch th, khoan to cc
ging. Nh mu th vo cc
ging. qua m, nh gi vng
c ch to thnh.
Phng php o
c
(Turbidometric
assay) [41]
Trong mi trng lng, mu
th cha bacteriocin s c ch
s pht trin ca vi sinh vt ch
th, lm cho s lng t bo vi
sinh vt ch th gim xung so
vi khi khng c mt mu th.
S lng t bo c nh gi
thng qua c ca mi
trng.
Cho mu th vo ng mi trng
lng cy vi sinh vt ch th.
qua m, o mt quang (OD
optical density) bc sng thch
hp, so vi ng i chng (khng
cho mu th). nh gi tc dng
c ch ca mu th i vi vi sinh
vt ch th.
-
Ph lc 4: Mt s qui trnh tinh ch bacteriocin ca LAB [57]
Bacteriocin Tinh ch S bc
Hiu sut (%)
Plantaricin ST31 Kt ta bng amoni sulfat Chit pha rn (Ct Sep-pak C18) RP HPLC
3 0,8
Sc k trao i cation 1 5,9
Nisin Z Sc k trao i ion nn m rng 1 90,0
Enterocin AS48 Trao i cation Sc k phn b pha o 2 24,3
Plantaricin C19 Hp ph / tch RP HPLC 2 15,0
Pediocin ACM Hp ph - Gii hp ph RP HPLC 2 40,4
Salivaricin Kt ta bng amoni sulfat Sc k phn b (Ct C18)
2 7,3
Pediocin PA-1 Kt ta bng ethanol in di tp trung ng in Siu lc
3 29,0
Lactococcin 3 41,0
Mesentericin
Y105
Sc k trao i cation Sc k phn b (Ct C18) HPLC
3 60,0
Sakacin A 3 10,0
Sakacin P 3 10,0
Enterocin A 3 66,0
Pediocin A-1 3 25,0
Divercin V41 3 10,0
Acidocin D20079 Kt ta bng amoni sulfat Sc k trao i cation Sc k tng tc k nc (Ct Octyl
Sepharose)
3 16,0
Lacticin Q Kt ta bng aceton Sc k trao i cation RP HPLC
3 64,0
Lactobin A Kt ta bng amoni sulfat Chit bng h dung mi chloroform : methanol RP HPLC
3 0,07
Divergicin M35 Sc k trao i cation Chit pha rn (Ct Sep-pak C18) RP HPLC
3 10,0
Enterocin CRL35 Kt ta bng amoni sulfat Lc phn t (Ct Biogel P-6) Sc k trao i cation HPLC
4 2,0
Bozacin 14 Kt ta bng amoni sulfat - Chit pha rn (Ct Sep-pak C18) RP HPLC
4 4,4
Mutacin B-Ny266 Sc k phn b (Ct C18) HPLC (x3) 4 1,0
Abp118 Kt ta bng amoni sulfat Sc k tng tc k nc Sc k trao i cation RP HPLC (Ct
C8 hoc C18)
4 6,4
Macedocin Kt ta bng amoni sulfat Sc k trao i anion Sc k trao i cation Sc k phn b
pha o Lc gel
5 1,6
Piscicocin CS526 Kt ta bng amoni sulfat Lc gel Sc k 5 7,0
-
trao i cation Sc k phn b (Ct C18) RP HPLC
Propionicin F Kt ta bng amoni sulfat Sc k trao i anion Sc k phn b pha o (x3)
5 0,5
Pediocin PD-1 Kt ta bng amoni sulfat Thm tch ng kh Chit bng methanol Chit bng
chloroform Trao i cation
6 34,0
-
Ph lc 5: Cc bacteriocin c tm thy Lactobacillus acidophilus [11]
Bacteriocin Chng vi khun Ph tc dng
Lactacin B L. acidophilus N Cc vi khun thuc h Lactobacillaceae
Lactacin F L. acidophilus
11088
Cc lactobacilli, Enterococcus faecalis
Acidophilucin
A
L. acidophilus
LAPT 1060
L. delbrueckii, L. helveticus
Acidocin
8912
L. acidophilus
TK8912
Lactobacillus v Lactococcus sp.
Acidocin B L. acidophilus M46 L. fermentum, Clostridium sporogenes
Acidocin A L. acidophilus
TK9201
Mt s LAB gn gi v Listeria monocytogenes
Acidocin
CH5
L. acidophilus CH5 Cc chi Lactobacillus, Bacillus, Micrococcus
v Corynebacterium
Acidocin
J1229
L. acidophilus JCM
1229
Lactobacillus v Lactococcus sp.
Acidocin
J1132
L. acidophilus JCM
1132
Lactobacillus sp.
Acidophilicin
LA-1
L. acidophilus LA-1 L. delbrucki, L. casei, L. jugurti
Acidocin
LF221A
L. acidophilus
LF221
Mt s LAB, B. cereus, Clostridium sp., L. innocua, S. aureus
Acidocin
LF221B
Acidophilin
801
L. acidophilus IBB
801
Mt s lactobacilli gn gi, E. coli v S. panama
Acidocin
D20079
L. acidophilus
DSM20079
Lactobacillus spp.
Acidocin
AA11
L. acidophilus
AA11
Salmonella, Shigella, E. coli, S. aureus, B.
cereus, B. subtilis
Acidocin 1B L. acidophilus
GP1B
Shigella, E. coli, P. aeruginosa, L.
monocytogenes, Yersinia enterocolitica
